H2-M3 is an MHC class Ib molecule which preferentially binds N-Formylated peptides. Since all prokaryotes initiate protein synthesis with N-formylated methionine, the peptide binding specificity of M3 is especially suited for presenting these unique microbial antigens to T cells. Consistent with this notion, mice infected with Listeria monocytogenes, generate CD8+ CTLs that recognize N-formylated Listeria peptides presented by M3. Recent studies have shown that M3-restricted T cells expand rapidly during primary Listeria infection, prior to the expansion of class Ia-restricted T cells. However, the expansion of M3-restricted T cells following secondary Listeria infection was rather limited compared with the vigorous recall response of class Ia-restricted T cells. The mechanisms underlying the distinct kinetics of the M3-restricted response are not clear and the significance of M3-restricted T cells in bacterial infection remains to be defined. This application seeks to understand how M3 presents bacterial antigens and how M3 contributes to shaping the T cell repertoire during bacterial infections. First, we will use biochemical and cell biology approaches to examine the structural requirements for controlling intracellular trafficking of M3 and to elucidate the pathway for presentation of both endogenous and exogenous listerial antigens by M3. Secondly, we will compare T cell development in the M3-deficient and control mice to determine whether M3 is responsible for selecting unique subset(s) of T cells. Adoptive transfer of naive and memory T cells from D7 transgenic mice, expressing TCR specific for M3/LemA complexes, will be performed to investigate whether M3-restricted T cells have requirements similar to class Ia-restricted T cells for the maintenance of the periphery T cell pools. Thirdly, we will infect M3-deficient, class Ia-deficient and control mice with Listeria to examine the relative contribution of class Ia-restricted and M3-restricted responses during Listeria infection, and to determine whether lack of an early M3 response could alter the kinetics and magnitude of class Ia-restricted response. Finally, we will extend our study to explore the functional role of M3-restricted T cells in immunity against Mycobaterium tuberculosis. Study of M3-restricted T cells responses against two distinct groups of intracellular bacteria would shed light on whether early and potent M3-restricted T cell response is unique to Listeria infection or is a general host defense mechanism against intracellular bacterial infection. Understanding the inter-relationship between class Ia-restricted and class Ib-restricted responses during the generation of specific immunity may facilitate the development of more effective vaccines.